Diuron lacks promoting potential in a rat liver bioassay

The promoting activity of the herbicide Diuron was evaluated in a medium-term rat liver carcinogenesis bioassay that uses as endpoint immunohistochemically identified glutathione S-transferase positive (GST-P+) foci. Male Wistar rats were allocated to the following groups: G1 to G6 were initiated for liver carcinogenesis by a single dose of diethylnitrosamine (DEN, 200 mg/kg) while groups G7 and G8 received only 0.9% NaCl (DEN vehicle). From the 2nd week animals were fed a basal diet (G1 and G7) or a diet added with Diuron at 125, 500, 1250, 2500 and 2500 ppm (G2 to G5 and G8, respectively) or 200 ppm Hexaclorobenzene (HCB; G6). The animals were submitted to 70% partial hepatectomy at the 3rd week and sacrificed at the 8th week. The herbicide did not alter ALT or creatinine serum levels. No conspicuous GST-P+ foci development was registered in non-initiated rats fed Diuron at 2500 ppm. While DEN-initiated animals fed Diuron at 1250 or 2500 ppm developed mild centrilobular hypertrophy, DEN-initiated HCB-fed animals showed severe liver centrilobular hypertrophy and significant GST-P+ foci development. These findings indicate that the medium-term assay adopted in this study does not reveal any liver carcinogenesis initiating or promoting potential of Diuron in the rat.     

Effects of progesterone on mammary carcinogenesis when various doses of DMBA were applied directly to rat mammae

To investigate the suggestion that progesterone acts directly on the mammary gland to inhibit carcinogenesis, doses of 500, 100 or 30 microgram of DMBA were applied directly to inguinal mammary glands of 50 d old rats which either received no other treatment or were injected with progesterone (3 mg/d) s.c. for 18 d before dusting the carcinogen. Progesterone pretreatment did not inhibit mammary carcinogenesis in rats dusted with 500 microgram or DMBA. When smaller doses of DMBA (100 microgram or less) were applied, hormone pretreatment markedly reduced carcinogenesis, the inhibitory effect being statistically significant in the group dusted with the smallest dose of carcinogen. As the dusting technique eliminated any observable systemic effects of the carcinogen, it is concluded that the results support the suggestion that progesterone acts directly on the mammary gland to inhibit carcinogenesis and that this effect can be over-ridden if a large enough dose of DMBA (somewhere between 100 and 500 micrograms) is applied. The importance of carcinogen dose to resulting tumour yield was clearly shown by the significant descending gradation in tumour incidences and gradual lengthening of average tumour latent periods in the 3 control groups with decreasing DMBA dose, as well as in the 3 hormone-pretreated groups.     

Identification of molecular phenotypes in canine mammary carcinomas with clinical implications: application of the human classification

Similarly to humans, canine mammary cancer represents a heterogeneous group in terms of morphology and biological behaviour. In the present study, we evaluated a series of canine mammary carcinomas based on a new human classification, initially based on gene expression profiling analysis. Similarly to human breast cancer, by using an immunohistochemistry surrogate panel based on five molecular markers [estrogen receptor, human epidermal growth factor receptor 2 (HER2), cytokeratin 5, p63 and P-cadherin], we were able to classify canine mammary carcinomas into four different subtypes: luminal A [estrogen receptor (ER)+/HER2-; 44.8%], luminal B (ER+/HER2+; 13.5%), basal (ER-/HER2- and a basal marker positive; 29.2%) and HER2 overexpressing tumours (ER-/HER2+; 8.3%). Luminal A-type tumours were characterised by lower grade and proliferation rate, whereas basal-type tumours were mostly high grade, high proliferative and positive for cytokeratin 5, p63 and P-cadherin. In addition, as in humans, basal subtype was significantly associated with shorter disease-free and overall survival rates, and we propose canine mammary carcinomas as a suitable natural model for the study of this particular subset of human carcinomas.     

Induction of cell proliferation, micronuclei and hyperdiploidy/polyploidy in the mammary cells of DDT- and DMBA-treated pubertal rats

The environmental estrogen, dichlorodiphenyltrichloroethane (DDT), and its metabolites have been implicated in the development of breast cancer through mechanisms that remain to be elucidated. It has been hypothesized that exposure to DDT and its metabolites, during critical periods of development, can contribute to an elevated risk for breast cancer in adults. In the present study, we have investigated the effect of o,p'-DDT on mammary gland cell proliferation and chromosomal alterations, in a rat mammary cancer model (commonly used to study human cancer), to gain insights into its potential role in the development of breast cancer. Twenty-one-day-old female Sprague-Dawley (SD) rats were administered o,p'-DDT, 7,12-dimethylbenz[a]anthracene (DMBA), genistein, DDT+DMBA, or DDT+DMBA+genistein, over a 14-day period. To determine changes in chromosome number and structure, we used the micronucleus assay as well as multicolor fluorescence in situ hybridization (FISH) region-specific DNA probes for rat chromosomes 4 and 19. Cell proliferation was evaluated using 5-bromo-2'-deoxyuridine (BrdU). Significant increases in BrdU-incorporated cells were seen in the rats treated with DDT+DMBA. Although micronucleus frequencies were somewhat elevated in several of the treatment groups, significant increases were not seen in any of them. Significant increases in numerical chromosomal aberrations were detected in all of the DDT- and DMBA-treated groups. Genistein significantly reduced BrdU incorporation and polyploidy in the DDT+DMBA-treated rats. These initial studies indicate that DDT and DMBA can induce cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that these agents can induce early events in mammary carcinogenesis.     

Positive two-stage carcinogenesis in female sprague-dawley rats using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and 12-o-tetradecanoylphorbol-13-acetate (TPA) as promotor

Summary In contrast to a previous report by Shubik, the validity of the 2-stage skin carcinogenesis experiment was demonstrated in the rat. The modified experiment was carried out in female Sprague-Dawley rats using intragastrically administered DMBA as a carcinogen and the topically applied phorbol ester TPA as a promoter.Seven groups of animals were used. Two groups were treated with TPA only, two groups were initiated only with DMBA, two further groups were both initiated and promoted, and one group served as a control. Each of the initiated/promoted groups or only initiated or promoted groups contained one sub-group in which the animals had been bilaterally ovarectomized prior to the experiment.Hyperplasia of the dorsal epidermis occurred only in the promoted and in the initiated/promoted groups. Tumors of the back skin were observed exclusively after initiation/promotion. Ovarectomy — leading to a prolonged survival time of the animals — seems to be crucial for the manifestation of malignant skin tumors. Initiation/promotion also gives rise to tumors of the forestomach, the small intestine, the liver and the colon. Tumors in other organs (especially in the mammary gland and the Zymbal gland) were also be observed after initiation alone.Dedicated to Professor Wilhelm Doerr on the occasion of his 65th anniversary     

Expression of cell cycle and apoptosis regulatory proteins in keratoacanthoma and squamous cell carcinoma

Some authors view keratoacanthoma (KA) as a variant of squamous cell carcinoma (SCC), while others consider it a separate entity that must be distinguished from SCC. Involution displayed by KA is an important difference between these two entities. It has been suggested that apoptosis plays a role in the involution process of KA, although the exact trigger for it remains unclear. A hundred and fifty specimens were included in this study, 30 cases for each of the following groups: normal skin (NS), proliferative keratoacanthoma (pKA), regressing keratoacanthoma (rKA), well-differentiated squamous cell carcinoma (wdSCC), and poorly differentiated squamous cell carcinoma (pdSCC). They were immunohistochemically examined for the expression of p53, Ki-67, bak, and bcl-2. Significantly higher p53 and Ki-67 expressions were observed in all tumor lesions examined as compared with NS. There was higher bak expression in KAs compared to NS and a significant reduction of bak expression in pdSCC together with a significant reduction of bak expression in SCCs compared to pKA. Bcl-2 expression was similar in NS and SCCs, but was lower in rKA. We found a significant positive correlation between p53 and Ki-67, p53 and Bak in NS and examined skin tumors. Lower bcl-2 expression in conjunction with higher bak expression in rKA suggests a possible role of these apoptosis-regulating proteins in tumor regression. In contrast to this finding, a steady level of bcl-2 expression in pdSCC combined with lower bak expression levels and a high proliferation rate could contribute to progression and aggressiveness in these tumors. Bak and p53 expression is a sun-related and age-dependent process in NS and skin tumors.     

Antitumor initiating potential of rosmarinic acid in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis

Oral cancer accounts for 40%-50% of all cancers in India. Tobacco and alcohol are the major etiological factors contributing to its pathogenesis. The aim of the present study was to explore the key mechanism behind the inhibitory effects of rosmarinic acid against 7,12-dimethylbenz(a)anthracene (DMBA)-induced oral carcinogenesis by evaluating the status of biochemical markers (lipid peroxidation, antioxidants, and detoxification enzymes) and immunoexpression patterns of p53 and bcl-2 proteins. Oral tumors were developed by painting the buccal pouches of golden Syrian hamsters with 0.5% DMBA in liquid paraffin 3 times a week for 14 weeks. We noticed 100% tumor formation in hamsters treated with DMBA alone, and the tumors were histopathologically confirmed as well-differentiated squamous cell carcinoma. Oral administration of rosmarinic acid (100 mg/kg body wt) to DMBA-treated hamsters completely prevented the tumor formation. In addition, rosmarinic acid significantly returned the status of biochemical and molecular markers to near normal range in DMBA-treated hamsters. The results of the present study suggest that rosmarinic acid suppresses oral carcinogenesis by stimulating the activities of detoxification enzymes, improves the status of lipid peroxidation and antioxidants, and downregulates the expression of p53 and bcl-2 during DMBA-induced oral carcinogenesis.     

Two relatively distinct patterns of ameloblastoma: an anti-apoptotic proliferating site in the outer layer (periphery) and a pro-apoptotic differentiating site in the inner layer (centre)

AIMS: This study was performed to determine the apoptotic behaviour of ameloblastomas by analysing the role of bcl-2 family proteins in ameloblastomas and the location of terminally apoptotic cells in the ameloblastoma epithelial tissues. METHODS AND RESULTS: For immunohistochemistry, tissue sections of 32 patients were treated with an antigen-retrieval METHOD: Primary antibodies against the apoptosis-related proteins, bcl-2, bcl-X, bax, and bak were applied. Besides immunohistochemistry, Western blotting and TUNEL were also performed. Most of the outer layer cells were predominantly stained by the bcl-2 antibody, while most of the inner layer cells were stained by antibodies against the apoptosis-modulating proteins, bax and bak. Among the bcl-2 family, bcl-2 was the most ubiquitously expressed protein in ameloblastomas, while bcl-X was expressed in the greatest concentrations. The major bcl-X protein was bcl-XL. Some of the inner layer cells entered the terminal apoptotic stage, which were revealed by TUNEL. The acanthomatous areas over-expressed the apoptosis-modulating proteins, especially bak. CONCLUSIONS: Ameloblastoma has much more apoptosis-inhibiting protein than the apoptosis-modulating protein. Ameloblastoma has two relatively distinct patterns, an anti-apoptotic proliferating site in the outer layer (periphery) and a pro-apoptotic differentiating site in the inner layer (centre). The acanthomatous area, which was stained strongly by bak antibody and contained numerous terminally apoptotic cells, was considered as the differentiated area.     

Influence of Strain and Sex on the Local Development of Mammary Tumors Induced by Direct Application of DMBA Powder to Rat Mammary Glands

In order to determine the influence of strain and sex on local carcinogenesis in rat mammary tissue, 1 mg of 7, 12 dimethylbenz(a)anthracene (DMBA) was dusted directly onto the exposed mammary gland of 30-day-old Long-Evans (L-E) rats and Sprague-Dawley (S-D) rats. The experiment was terminated 28 weeks after application of the carcinogen. Tumors measuring between 1 and 2 cm in diameter were harvested from female L-E rats with high frequency (85%) and long latency (mean: 23.7 weeks after DMBA dusting), and from female S-D rats with extremely high frequency (98%) and short latency (16.7 weeks). Male rats of both strains were almost identically much less susceptible to DMBA (L-E; 55%, 25.0 weeks, S-D; 53%, 23.9 weeks). Ovariectomized S-D (47%, 24.9 weeks) and orchiectomized S-D (30%, 24.8 weeks) rats, which were gonadectomized at 30 days of age, respectively, were also much less susceptible. A variety of histologies, mostly malignant epithelial, mesenchymal or mixed tumors, were noted in each group. The carcinomatous response in the mammary tissue was much higher in female S-D (96%) than in female L-E (50%) rats, and very low in male and gonadectomized rats (10-20%). In contrast, the sar-comatous response in the mammary tissue was moderate in female and male L-E and male S-D (43-50%) rats, and low in the other groups (15-29%). Acta Pathol Jpn 40: 9–13, 1990.     

Influence of strain and sex on the local development of mammary tumors induced by direct application of DMBA powder to rat mammary glands

In order to determine the influence of strain and sex on local carcinogenesis in rat mammary tissue, 1 mg of 7,12-dimethylbenz(a)anthracene (DMBA) was dusted directly onto the exposed mammary gland of 30-day-old Long-Evans (L-E) rats and Sprague-Dawley (S-D) rats. The experiment was terminated 28 weeks after application of the carcinogen. Tumors measuring between 1 and 2 cm in diameter were harvested from female L-E rats with high frequency (85%) and long latency (mean: 23.7 weeks after DMBA dusting), and from female S-D rats with extremely high frequency (98%) and short latency (16.7 weeks). Male rats of both strains were almost identically much less susceptible to DMBA (L-E; 55%, 25.0 weeks, S-D; 53%, 23.9 weeks). Ovariectomized S-D (47%, 24.9 weeks) and orchiectomized S-D (30%, 24.8 weeks) rats, which were gonadectomized at 30 days of age, respectively, were also much less susceptible. A variety of histologies, mostly malignant epithelial, mesenchymal or mixed tumors, were noted in each group. The carcinomatous response in the mammary tissue was much higher in female S-D (96%) than in female L-E (50%) rats, and very low in male and gonadectomized rats (10-20%). In contrast, the sarcomatous response in the mammary tissue was moderate in female and male L-E and male S-D (43-50%) rats, and low in the other groups (15-29%).     

Modifying effects of antioxidants on chemical carcinogenesis

Studies were made on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats, mice, and hamsters and the effect of the antioxidants BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), sodium L-ascorbate (SA), ascorbic acid (AA), sodium erythorbate (SE), propyl gallate (PG), and alpha-tocopherol, on two-stage chemical carcinogenesis in rats initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 1,2-dimethylhydrazine (DMH), diethylnitrosamine (DEN), 7,12-dimethylbenz(a)anthracene (DMBA), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-ethyl-N-hydroxyethylnitrosamine (EHEN), or N-methylnitrosourea (MNU). BHA clearly induced squamous cell carcinomas in both the rat and hamster forestomach. The tumorigenic action of crude BHA on the forestomach is largely due to 3-tert-BHA. In two-stage chemical carcinogenesis, BHA promoted MNNG or MNU-initiated forestomach and BBN- or MNU-initiated urinary bladder carcinogenesis and inhibited DEN- or EHEN-initiated liver and DMBA-initiated mammary carcinogenesis. BHT demonstrated promotion potential for urinary bladder and MNU-initiated thyroid carcinogenesis and inhibited DMBA-initiated ear duct carcinogenesis. EQ promoted EHEN-initiated kidney carcinogenesis and inhibited DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. SA promoted forestomach and urinary bladder carcinogenesis and SE likewise enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear duct carcinogenesis. No effects of any of the antioxidants on glandular stomach carcinogenesis were found. The results clearly demonstrated that antioxidants have different effects (promoting or inhibitory influences) depending on the organ studied and suggest the importance of a whole body approach to their investigation.     

Modifying effects of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids Re-80, Am-580 and Am-55P in a two-stage carcinogenesis model in female rats

Effects of dietary administration of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids 4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (Re-80); 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (Am-580); and 6-[(3,5-di-tert-butylphenyl) carbamoyl]nicotinic acid (Am-55P) were examined using a two-stage rat carcinogenesis model. A total of 190 female SD rats was treated sequentially with 1,2-dimethylhydrazine (DMH, s.c.); 7,12-dimethylbenz(a)anthracene (DMBA, i.g.); and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN, in the drinking water) during the first three weeks (DDD-initiation), and an additional 60 rats received the vehicle alone (non-initiation). One week after the completion of the initiation period, they were divided into nine groups and administrated Re-80 (at dose levels of 1.0 or 0.4 ppm), Am-580 (20 or 4 ppm), Am-55P (20 ppm), ACA (100 ppm), all-trans-retinoic acid (10 or 2 ppm) or no supplement in the diet for 33 weeks, until survivors were euthanatized at week 37 weeks. After DDD-initiation, all-trans-retinoic acid at the high dose delayed the development of mammary tumors. The multiplicity of colon tumors in the group fed Am-55P and the incidences of nephroblastomas with ACA or Am-580 were decreased as compared with the control values, but the other chemicals had no modifying effects on tumor development in any organs. Thus, among ACA and the novel synthetic retinoids tested, only Am-55P showed a weak inhibitory effect on a neoplasm of general interest under the present experimental conditions.     

Genistein and daidzein, in combination, protect cellular integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in Sprague-Dawley rats

The status of glycoconjugates (protein bound hexose, hexosamine, sialic acid and fucose) in plasma or serum serve as potential biomarkers for assessing tumor progression and therapeutic interventions. Aim of the present study was to investigate the protective effect of two major soy isoflavones, genistein and daidzein, in combination on the status of glycoconjugates in plasma, erythrocyte membrane and mammary tissues during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in female Sprague-Dawley rats. A single subcutaneous injection of DMBA (25 mg rat(-1)) in the mammary gland developed mammary carcinoma in female Sprague-Dawley rats. Elevated levels of plasma and mammary tissue glycoconjugates accompanied by reduction in erythrocyte membrane glycoconjugates were observed in rats bearing mammary tumors. Oral administration of genistein + daidzein (20 mg + 20 mg kg(-1) bw/day) to DMBA treated rats significantly (p< 0.05) brought back the status of glycoconjugates to near normal range. The present study thus demonstrated that genistein and daidzein in combination protected the structural integrity of the cell surface and membranes during DMBA-induced mammary carcinogenesis.     

The effects of neonatal androgenization on mammary gland mitotic rate and susceptibility of carcinogen-induced mammary dysplastigenesis and tumorigenesis in LEW/Mai rats.

The influence of neonatal testosterone propionate treatment (androgenization) on mammary gland mitotic rate (MR) and susceptibility to 7,12-dimethylbenz(alpha)anthracene (DMBA) carcinogenesis was studied in female LEW/Mai rats. Mammary gland MR in androgenized rats was significantly lower than MR in normal rats at all ages studied. Treatment of androgenized rats with DMBA resulted in a significant increase in mammary gland MR in comparison with untreated androgenized rats. MR in DMBA-treated androgenized rats was similar to MR in DMBA-treated normal rats at most intervals after the introduction of the carcinogen. Although mammary epithelial MR in androgenized rats was significantly lower than that of normal rats of comparable age, no evidence of a decrease in susceptibility of mammary epithelium in androgenized rats to DMBA carcinogenesis was found. Instead, androgenized rats had a higher incidence of DMBA-induced mammary dysplasias, with no change in their morphologic or histologic features, than did normal rats; and there was no change in the incidence, latency, or histopathologic appearance of DMBA-induced mammary tumors in androgenized versus normal rats.     

Susceptibility of the mammary gland to carcinogenesis. III. The cell of origin of rat mammary carcinoma.

The rat mammary gland epithelium is composed of three cell types: dark cells (DCs), intermediate cells (ICs), and a layer of myoepithelial cells (MCs), which are evenly distributed along the mammary gland tree in rather constant proportions. The present study was carried out for a determination of the effect of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) on the distribution and proliferative activity of these cell populations. The proportion and the DNA labeling index (DNA-LI) of each cell type were determined in terminal end buds (TEBs), terminal ducts (TDs), alveolar buds (ABs), and alveoli of the normal Sprague-Dawley rat mammary gland and in intraductal proliferations (IDPs) and carcinomas removed at selected intervals after DMBA administration. DMBA-induced changes in cell distribution were limited to TEBs and TDs, whereas ABs and alveoli were unaffected. The alterations consisted in an increment in ICs from 11% in TEBs and TDs to 90% in tumors and a decrease in DCs from 77% in TEBs and TDs to 7% in tumors. MCs were relatively unaffected. The DNA-LI of DCs, which in the normal gland TEB was 14%, was depressed by DMBA to 6%, whereas the DNA-LI of ICs remained unchanged from the basal level of 40% during the process of carcinogenesis. The progressive increment in number of ICs with a steady DNA-LI suggested that the IC is the target cell of the carcinogen and the cell of origin of mammary carcinomas.     

Induction of pulmonary carcinogenesis in Wistar rats by a single dose of 9, 10 Dimethylbenz(a)anthracene (DMBA) and the chemopreventive role of Diclofenac

Objectives

To evaluate the chemopreventive efficacy of Diclofenac, a preferential cyclooxygenase-2 (COX-2) inhibiting non steroidal anti-inflammatory drug (NSAID) in the 9, 10 Dimethylbenz(a)anthracene (DMBA) induced experimental lung carcinogenesis.

Methods

Animals were divided into 4 groups. Control group received normal saline intratratracheally. DMBA group was given DMBA (20 mg/kg of body weight) in the similar manner. DMBA + Diclofenac group was given daily oral dose of Diclofenac (8 mg/kg of body weight) in addition to DMBA while the last group received Diclofenac only. Animals were sacrificed after 24 weeks. COX-2 expression was studied by immunohistochemistry (IHC) and Western immunoblotting. For apoptosis study DNA fragmentation on agarose gel and florescent staining of alveolar macrophages were done.

Results

The incidence and burden of tumor were reduced by the Diclofenac treatment. Diclofenac caused the reduction in the COX-2 levels which were increased in the DMBA treated group. It also caused the induction of apoptosis as seen by both techniques.

Conclusion

From all these results it can be concluded that Diclofenac might have a chemopreventive role for lung carcinogenesis which is mediated by suppression of COX-2 enzyme and induction of apoptosis.     

Cytochemical investigation of glucose-6-phosphate dehydrogenase activity in rat mammary tissue

Glucose-6-phosphate dehydrogenase (G6PD) activity in epithelial cells from six normal, eight lactating and 21 DMBA tumour bearing rat mammary tissues was investigated using techniques of quantitative cytochemistry. G6PD promoted H+ production was quantified under atmospheres of N2 and O2 in frozen sections from rat mammary tissue in the presence and absence of a H+ acceptor (Total H+ and Type I H+). There was a considerable overlap between the three tissue types in values for Total H+ measured under N2 or O2. However, maximum H+ production in lactating and DMBA tumour tissue took longer to achieve in O2 than in N2 (16-20 min vs 6-8 min). In normal tissue maximum production of Total H+ was not achieved until 45-50 min and the rate of reaction was similar in N2 and in O2. Type I H+ measured in N2 did not vary significantly between DMBA tumours and lactating tissue but under O2 was only present in DMBA tumour, being undetectable in both normal and lactating tissue. The results demonstrate that despite the overlap in G6PD activities between the tissues tested, the techniques of quantitative cytochemistry can provide a functional assay differentiating between non-malignant and malignant breast tissue in the rat.     

Cytochemical investigation of glucose-6-phosphate dehydrogenase activity in rat mammary tissue.

Glucose-6-phosphate dehydrogenase (G6PD) activity in epithelial cells from six normal, eight lactating and 21 DMBA tumour bearing rat mammary tissues was investigated using techniques of quantitative cytochemistry. G6PD promoted H+ production was quantified under atmospheres of N2 and O2 in frozen sections from rat mammary tissue in the presence and absence of a H+ acceptor (Total H+ and Type I H+). There was a considerable overlap between the three tissue types in values for Total H+ measured under N2 or O2. However, maximum H+ production in lactating and DMBA tumour tissue took longer to achieve in O2 than in N2 (16-20 min vs 6-8 min). In normal tissue maximum production of Total H+ was not achieved until 45-50 min and the rate of reaction was similar in N2 and in O2. Type I H+ measured in N2 did not vary significantly between DMBA tumours and lactating tissue but under O2 was only present in DMBA tumour, being undetectable in both normal and lactating tissue. The results demonstrate that despite the overlap in G6PD activities between the tissues tested, the techniques of quantitative cytochemistry can provide a functional assay differentiating between non-malignant and malignant breast tissue in the rat.     

Studies on mammary carcinogenesis induced by a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, in mice and rats

Ten heterocyclic amines (HCAs) that are produced by heating amino acids, proteins, or proteinaceous food such as fish and meat were examined for carcinogenicity in rats and mice. Three of them, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), have been shown to induce mammary cancer in female F344 and/or SD rats, but none of the HCAs induced mammary cancer in CDF(1) mice. This report reviews our recent studies on mammary carcinogenesis of PhIP in various strains of mice and on the roles of genomic instability in the rat mammary carcinogenesis of PhIP. We demonstrated that the survival time from mammary adenocarcinomas was shorter in PhIP-treated BALB/c mice than that in the untreated control, and with a significantly higher incidence in the C.B-17 strain of mice compared with that of the control. To clarify mechanisms of mammary carcinogenesis, we examined genomic instability in rat mammary cancer induced by PhIP. Mammary cancers were induced in F344 x SD F(1) rats harboring the lacI transgene, and two cell lines were established from two adenocarcinomas. They showed a greater than 10-fold higher frequency of spontaneous mutations than that of the primary culture of normal mammary epithelial cells, in the lacI transgene and the hprt endogenous gene during cell replication. Nucleotide sequencing revealed that almost all types of mutations were increased, with a remarkable increase of A:T --> C:G mutation. This genomic instability was not attributed either to alterations of mismatch-repair enzymes or to p53. These mutational characteristics were also observed in the original tumors. Single-nucleotide instability (SNI) might be implicated in the mammary cancer induced by PhIP.     

A spontaneous high-grade undifferentiated mammary carcinoma in a seven-week-old female rat

The present work describes a rare case of a spontaneous high-grade carcinoma in a seven-week-old Sprague-Dawley female rat that had been included in the control group of an assay of mammary carcinogenesis. The mass was detected at 50 days of age, it grown quickly and the animal was humanely sacrificed eight days later. The tumor was located in the left cervical region, in the vicinity of the left submandibular and sublingual glands. It was soft and reddish and had several dens with a bloody content. The tumor was PAS negative and exhibited immunostaining for ER-α. The histopathologic and immunohistochemical data are suggestive of a high-grade carcinoma from mammary gland. It was the first report of a spontaneous mammary tumor in such a young rat.